In this project we will further investigate two key observations made in the past funding period: 1) P-selectin[unreadable] induces the formation of pro-coagulant microparticles (MPs) from leukocytes, and 2) activation of the platelet[unreadable] metalloproteinase ADAM17 (TACE) leads to platelet clearance.[unreadable] In the first aim, we will study the cell biology of MP production induced by IPS or a recombinant P-selectin[unreadable] molecule (P-sel-lg), in vitro and in vivo. The working hypothesis is that MPs are released from the plasma[unreadable] membrane through a budding process that employs cellular machinery used for the formation of multivesicular[unreadable] endosomes and for retrovirus budding. We will study the importance of ubiquitination and the cellular[unreadable] cytoskeleton in this process. The effect of shear stress on MP production and the adhesion molecules[unreadable] involved in MP recruitment to thrombi will be examined together with Project 3. With Project 2, we will[unreadable] characterize MPs produced in mice over-expressing soluble P-selectin (deltaCT-P-sel mice) for their cytoskeleton[unreadable] and surface proteins. MP generation will be studied in cytoskeleton-mutant mice (filamin chimeras, gelsolin -/-).[unreadable] The role of the cytoplasmic domain of tissue factor (TF) in TF targeting to MPs and in the regulation of its procoagulant[unreadable] activity will be studied in mice expressing TF lacking the CT domain (deltaCT-TF mice).[unreadable] In the second aim, we propose to study the role of TACE in platelet biology. We hypothesize that the signaling[unreadable] pathways that regulate the proteolytic activity of TACE are important for both platelet function in hemostasis[unreadable] and in the regulation of platelet clearance. We will examine the intracellular processing and the sub-cellular[unreadable] localization of TACE upon platelet activation or mitochondrial injury. It is our hypothesis that, in platelets,[unreadable] TACE translocation to the plasma membrane coincides with its activation. We will address the signaling[unreadable] pathways from mitochondrial injury to TACE activity with a focus on AMP-activated protein kinase (AMPK), a[unreadable] metabolic stress-sensing kinase, and on p38 MAP kinase. In in vitro and in vivo models of arterial and venous[unreadable] thrombosis, we will determine the role of TACE and the kinases involved in its activation in thrombus[unreadable] formation. For these studies we will use genetically modified mice and inhibitors of the activating kinases.[unreadable] In the third aim, we will address problems directly relevant to transfusion biology: how are damaged[unreadable] (mitochondrial injury or aging) platelets cleared from circulation, and how to improve the efficacy of platelet[unreadable] transfusions by adding TACE inhibitors to platelet concentrates and/or co-infusing P-sel-lg. We will test the[unreadable] hypotheses that TACE regulates platelet clearance by shedding of GPIba and/or clustering of GPIb-V-IX[unreadable] complexes, and that P-selectin, by producing pro-coagulant MPs, would enhance hemostasis in[unreadable] thrombocytopenic mice.